Systemic Toxicity

Poisoning is a leading cause of injury-related death in the United States. As such, regulatory agencies require systemic toxicity testing on all potentially hazardous substances so that appropriate warning labels may be applied. Assessment of the systemic toxicity potential of a test material has traditionally been evaluated using animal tests to determine an LD50 value (the median lethal dose of a test material that kills 50% of test animals). However, the in vitro Neutral Red Uptake cytotoxicity assays in Balb/c 3T3 and NHEK cells are now being utilized to estimate the systemic toxicity of chemicals and to reduce the number of animals required to perform LD50 determinations.

Ekwall et al. (ATLA 17:83-100, 1989) proposed that approximately 80% of chemical-induced systemic toxicity is the result of disruption of basic cellular processes common to most cell types in the body, and that systemic toxicity for many chemicals could be estimated in in vitro cultures. Healthy mammalian cells when maintained in culture continuously proliferate. A toxic chemical, regardless of site or mechanism of action, will interfere with this process and result in a reduction in viable cells relative to untreated controls.

The viability of cells can be assessed using a neutral red uptake (NRU) endpoint. A decrease in the uptake of neutral red dye in treated cell cultures following a test chemical exposure is used to determine relative toxicity. Neutral Red (NR) is a weak cationic dye that readily diffuses through cell membranes and accumulates in cellular lysosomes. Once in the acidic environment of the lysosome, the NR is oxidized, becoming positively charged and trapped within the lysosome. Unless the cell or lysosome is damaged, the red dye remains trapped. Alterations to the cell membrane (caused by toxicity of the test material) are generally irreversible, resulting in the loss of the NR from the lysosome.

IIVS was the lead laboratory in the validation of the 3T3 and NHEK NRU Cytotoxicity Assays for Estimating Starting Doses for Rodent LD50 Values. The validation was funded by the European Center for Validation of Alternative Methods (ECVAM) and the National Institute of Environmental Health Sciences (NIEHS).